Apparatus and methods for syntehsizing biopolymers

ABSTRACT

The present disclosure provides an apparatus for synthesizing a biopolymer, a method for preparing an apparatus for synthesizing a biopolymer, and a method of synthesizing a biopolymer. The apparatus comprises (a) a substrate comprising a top surface and a plurality of wells, wherein each of the plurality of wells comprises a first electrode disposed on the bottom of the well and a linker attached to the sides of the well; and (b) a fluidic chamber system disposed on the top surface of the substrate.

FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under R21 HG009363awarded by the National Science Foundation and FA9550-16-1-0052 awardedby the Air Force of Scientific Research (AFOSR). The government hascertain rights in the invention.

SUMMARY

The present disclosure provides an apparatus for synthesizing abiopolymer. The apparatus comprises (a) a substrate comprising a topsurface and a plurality of wells, wherein each of the plurality of wellscomprises a first electrode disposed on the bottom of the well and alinker attached to the sides of the well; and (b) a fluidic chambersystem disposed on the top surface of the substrate.

The present disclosure also provides a method for preparing anapparatus. The method comprises (a) forming a plurality of wells on asubstrate; (b) applying a first electrode to the bottom of the well; (c)attaching a linker to the sides of the well; and (d) affixing a fluidicchamber onto the substrate.

The present disclosure also provides a method of synthesizing anoligonucleotide. The method comprises (a) providing an apparatus asherein described; (b) introducing a solution comprising a firstnucleotide phosphoramidite monomer into the well, wherein the firstphosphoramidite monomer comprises a 5′-protecting group, an acidsensitive protecting group and optionally a base sensitive protectinggroup, and wherein the first phosphoramidite monomer reacts with thelinker attached to the side walls of the well to form a linkednucleotide with a phosphite triester; (c) removing the solution fromstep (b) from the well; (d) introducing a solution comprising a cappingreagent into the well, wherein the capping reagent reacts with anyunreacted linker from step (b) to form a capped linker; (e) removing thesolution from step (d) from the well; (f) introducing a solutioncomprising an oxidant into the well, wherein the oxidant converts thephosphite triester of the linked nucleoside to a phosphate triester; (g)removing the solution from step (f) from the well; (h) introducing asolution comprising a first redox reagent into the well, wherein a localpotential is applied to the electrode inside the selected well such thatthe electrochemical reaction controlled by the potential induces theremoval of the 5′-protecting group; in one of the embodiment suchelectrochemical reaction involves the oxidation of hydroquinone whichdecreases pH locally inside the well; (i) removing the solution fromstep (h) from the well; (j) repeating steps (b) through (i) tosynthesize a protected oligonucleotide; and (k) introducing a solutioncomprising a second deprotecting reagent into the well, wherein thesecond deprotecting agent removes the protecting groups on theoligonucleotide.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A shows the apparatus design according to a first embodiment ofthe disclosure. FIG. 1B shows an exploded view of an array ofindividually addressable reaction sites connected to an external contactpad. FIG. 1C shows a side view of the reaction site.

FIG. 2A shows the apparatus design according to a second embodiment ofthe disclosure in which the top surface of the apparatus, excluding thereaction sites (wells), comprises a passivation layer. FIG. 2B shows anexploded view of an array of individually addressable reaction sitesconnected to an external contact pad. FIG. 2C shows a side view of thereaction site after application of a passivation layer. FIG. 2D shows aside view of the reaction site in which the top surface of theapparatus, excluding the reaction sites, comprises a passivation layer.

FIG. 3A-FIG. 3D show a method of preparing an apparatus according to afirst embodiment of the disclosure. FIG. 3A shows the application of aphoto resist onto the surface of a substrate to define a well. FIG. 3Bshows etching of the substrate to create a well having a defined depth.FIG. 3C shows the application of a first electrode to the bottom of thewell. FIG. 3D shows the removal of the photo resist.

FIG. 4A-FIG. 4E show a method of preparing an apparatus according to asecond embodiment of the disclosure. FIG. 4A shows the application of apassivation layer on a substrate. FIG. 4B shows the application of aphoto resist onto the surface of a substrate to define a well. FIG. 4Cshows etching of the substrate to create a well having a defined depth.FIG. 4D shows the application of a first electrode to the bottom of thewell. FIG. 4E shows the removal of the photo resist.

FIG. 5A-FIG. 5C shows the fabrication process of passivation layeraccording to an embodiment of the disclosure. FIG. 5A shows theapplication of a photo resist onto the surface of a substrate to sealall the trenches. FIG. 5B shows the exposure and post exposure bakingproses to define the passivation layer. FIG. 5C shows the developmentand removal of unexposed resist such that all metal surfaces except thatin the reaction sites are sealed (in FIG. 5 the metal cross sectionrepresent the connection wires that is sealed by this passivation as inFIG. 2C, for all the other cases, the metal cross section represent thereaction sites that is exposed as in FIG. 2D).

FIG. 6 shows the attachment of a linker to the sides of the wellaccording to an embodiment of the disclosure.

FIG. 7 shows an apparatus according to a third embodiment of thedisclosure.

FIG. 8 shows a summary of the oligonucleotide synthesis cycle.

FIG. 9A-FIG. 9H show a method of synthesizing oligonucleotides accordingto an embodiment of the disclosure. FIG. 9A shows the first step inwhich a phosphoramidite monomer is introduced into the well. The monomerreacts with the linker attached to the side walls of the wells to form anucleoside with a phosphite triester linked to the side walls of thewell. FIG. 9B shows the second step of capping in which unreacted linkeris capped with an unreactive group. FIG. 9C shows the third step ofoxidation in which the phosphite triester is converted to a phosphatetriester. FIG. 9D shows the fourth step of electrochemically-controlleddeprotection in which the 5′-protecting group is removed. FIG. 9E showsthe fifth step in which a second phosphoramidite monomer is introducedinto the well. The second phosphoramidite monomer reacts with 5′-OH ofthe linked nucleotide to form a dinucleotide phosphite triester. FIG. 9Fshows the sixth step of oxidation in which the phosphite triester isconverted to a phosphate triester. FIG. 9G shows the seventh step ofdeprotection in which the protecting groups on from the bases andphosphates are removed. FIG. 9H shows the final step of deprotection inwhich the cyanoethyl groups from the nucleotide are removed

FIG. 10A shows an apparatus according to an embodiment of the disclosureand used in Example 1. FIG. 10B shows the epifluorescence image ofnegative control test. FIG. 10C shows the fluorescent intensity measuredfrom FIG. 10B, indicating the background fluorescent intensity.

FIG. 11A shows the epifluorescence image of positive control test. FIG.11B shows the fluorescent intensity measured from FIG. 11A, indicatingthe fluorescent intensity of the positive control test.

FIG. 12A shows the epifluorescence image of electrochemical deprotectionresult near activated electrode FIG. 12B shows the fluorescent intensitymeasured from FIG. 12A, indicating the fluorescent signal is comparableto positive control test.

FIG. 13 shows an apparatus according to an embodiment of the disclosureand used in Example 2.

FIG. 14A shows the epifluorescent image of electrode that is activatedfor 1 second. FIG. 14B shows the fluorescent intensity measured fromFIG. 14A. The relatively low intensity indicates insufficient protonconcentrating.

FIG. 15A shows the epifluorescent image of electrode that is activatedfor 5 second. FIG. 15B shows the fluorescent intensity measured fromFIG. 15A. The fluorescent intensity gradient indicates the diffusion ofthe electrochemically-generated proton.

FIG. 16A shows the epifluorescent image of electrode that is activatedfor 10 second. FIG. 16B shows the fluorescent intensity measured fromFIG. 16A. The globally lighten up image indicates electrochemicallygenerated proton may diffuse to inactivated area following the flowingdirection of the reagents.

FIG. 17 shows a schematic diagram of a 12 mer DNA synthesis verified byhybridization with a fluorescent labeled complimentary DNA.

FIG. 18 A-18D show the results of the localized electrochemicalsynthesis of 12 mer DNA within the side walls of the reaction sitesverified by hybridization with a fluorescent labeled complimentary DNA.FIG. 18 A shows the fluorescent optical image of electrochemicallyactivated reaction site. FIG. 18 B shows the fluorescent intensitymeasured from FIG. 18 A. FIG. 18 C shows the fluorescent optical imageof another electrochemically activated reaction site. FIG. 18 D showsthe fluorescent intensity measured from FIG. 18 C.

FIG. 19A and FIG. 19B show the results of negative control indicatingthat no obvious fluorescence is observed. The background intensity is160.

FIG. 20A and FIG. 20B show the results of a positive control in which a12 mer DNA with an identical sequence was synthesized throughconventional chemical activation on the side walls of the reactionsites. The signal/noise ratio is about 1.6, comparable withelectrochemical activation. The overall higher background compared toprevious electrochemical activation sample is due to the immersion oilused with the objective lens that has higher auto fluorescence level.

DETAILED DESCRIPTION

In various embodiments, the invention includes some or all of thefollowing:

1. An apparatus for synthesizing a biopolymer, the apparatus comprising:

-   -   (a) a substrate comprising a top surface and a plurality of        wells, wherein each of the plurality of wells comprises a first        electrode disposed on the bottom of the well and a linker        attached to the sides of the well; and    -   (b) a fluidic chamber system disposed on the top surface of the        substrate.

2. The apparatus according to the above 1, wherein the distance betweena first well and a second well is about 1 to about 200 μm.

3. The apparatus according to the above 1, wherein the diameter of thewells is about 1 to about 500 μm.

4. The apparatus according to the above 1, wherein the substrate isselected from glass, sputtered SiO₂, PECVD, SiO₂ and the like.

5. The apparatus according to the above 1, wherein the depth of the wellis from about 500 nm to about 500 μm.

6. The apparatus according to the above 1, wherein the depth of the wellis from about 500 nm to 10 μm.

7. The apparatus according to the above 1, wherein the first electrodecomprises palladium, platinum, gold, or carbon.

8. The apparatus according to the above 1, wherein the linker comprisesa hydroxy functionalized silane.

9. The apparatus according to the above 1, wherein the linker comprisesan amine or thiol functionalized silane.

10. The apparatus according to the above 1, wherein the linker is(N-(3-triethoxysilylpropyl)-4-hydroxybutyramide.

11. The apparatus according to the above 1, wherein the fluidic chambersystem comprises polydimethylsiloxane (PDMS).

12. The apparatus according to the above 1, wherein the fluid chambersystem has a height of about 50 μm to 10 mm.

13. The apparatus according to the above 1, wherein the fluid chambersystem comprising a second electrode.

14. The apparatus according to the above 13, wherein the secondelectrode comprises palladium, platinum, gold, or carbon.

15. The apparatus according to the above 1, wherein the fluid chambersystem further comprises a system for introducing and removing liquidsfrom the well.

16. A method for preparing an apparatus, comprising

-   -   a. forming a plurality of wells on a substrate;    -   b. applying a first electrode to the bottom of the well;    -   c. passivating the surface of the connections of the electrodes;    -   d. attaching a linker to the sides of the well; and    -   e. affixing a fluidic chamber onto the substrate.

17. The method of the above 16, wherein step (a) comprises:

-   -   i. applying a photo resist onto the surface of the substrate to        define the plurality of wells; and    -   ii. etching the substrate to create the plurality of wells.

18. The method of the above 16, wherein the substrate is selected fromglass, sputtered SiO₂, PECVD, SiO₂ and the like.

19. The method of the above 16, wherein the first electrode comprisespalladium, platinum, gold, or carbon.

20. The method of the above 16, wherein step (b) comprises depositing ametal selected from the group consisting of palladium, platinum, goldany other metals that is suitable for electrochemical control in thedeprotection reagent;

21. The method of the above 17, wherein step (b) further comprisesremoving the photo resist.

22. The method of the above 16, wherein step (c) comprises immersing thesubstrate from step (b) into a linker solution for a period of time; andremoving the linker solution.

23. The method of the above 16, wherein the fluid chamber systemcomprises a second electrode.

24. The method of the above 23, wherein the second electrode comprisespalladium, platinum, gold, carbon or other conducting materials suitablefor electrochemical control in the deprotection reagent.

25. The method of the above 16, wherein the fluid chamber system furthercomprises a system for introducing and removing liquids from the well.

26. A method of synthesizing an oligonucleotide, the method comprising

-   -   (a) providing an apparatus according to any one of the above 1        to 15;    -   (b) introducing a solution comprising a first nucleoside        phosphoramidite monomer into the well, wherein the first        phosphoramidite monomer comprises a 5′-protecting group, an acid        sensitive protecting group and optionally a base sensitive        protecting group, and wherein the first phosphoramidite monomer        reacts with the linker attached to the side walls of the well to        form a linked nucleoside through a phosphite triester;    -   (c) removing the solution from step (b) from the well;    -   (d) introducing a solution comprising a capping reagent into the        well, wherein the capping reagent reacts with any unreacted        linker from step (b) to form a capped linker;    -   (e) removing the solution from step (d) from the well;    -   (f) introducing a solution comprising an oxidant into the well,        wherein the oxidant converts the phosphite triester of the        linked nucleoside to a phosphate triester;    -   (g) removing the solution from step (f) from the well;    -   (h) introducing a solution comprising a first deprotecting        reagent into the well, wherein the deprotecting reagent is        electrochemically activated and removes the 5′-protecting group;        in one embodiment, this activation involves the oxidation of        hydroquinone to locally generate protons inside the well.    -   (i) removing the solution from step (h) from the well;    -   (j) repeating steps (b) through (i) to synthesize a protected        oligonucleotide; and    -   (k) introducing a solution comprising a second deprotecting        reagent into the well, wherein the second deprotecting agent        removes the protecting groups on the oligonucleotide.

27. The method of the above 26, wherein the capping reagent is aceticanhydride.

28. The method of the above 26, wherein the capped linker comprises anacetate group.

29. The method of the above 26, wherein the oxidant is(1S)-(+)-(10-camphorsulfonyl)-oxaziridine (CSO).

30. The method of the above 26, wherein the first deprotecting reagentremoves a 5′-trityl group.

31. The method of the above 30, wherein the first deprotecting reagentis electrochemically generated acid.

32. The method of the above 26, wherein the second deprotecting reagentremoves the phosphate protecting group.

33. The method of the above 26, wherein the second deprotecting reagentremoves the acid sensitive protecting group.

34. The method of the above 31-33, wherein the second deprotectingreagent is ethylenediamine.

DEFINITIONS

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as those commonly understood by one of ordinaryskill in the art to which this invention belongs. Although methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present disclosure, suitable methods andmaterials are described below. The materials, methods and examples areillustrative only, and are not intended to be limiting. Allpublications, patents and other documents mentioned herein areincorporated by reference in their entirety.

Throughout this specification, the word “comprise” or variations such as“comprises” or “comprising” will be understood to imply the inclusion ofa stated integer or groups of integers but not the exclusion of anyother integer or group of integers.

The term “a” or “an” may mean more than one of an item.

The terms “and” and “or” may refer to either the conjunctive ordisjunctive and mean “and/or”.

The term “about” means within plus or minus 10% of a stated value. Forexample, “about 100” would refer to any number between 90 and 110.

Apparatus

The present disclosure provides an apparatus for synthesizing abiopolymer. The biopolymer is an oligonucleotide in one embodiment and aprotein in a second embodiment. In one aspect, the biopolymer is DNA. Ina second aspect, the biopolymer is RNA. In a third aspect, thebiopolymer is a peptide.

The apparatus comprises (a) a substrate comprising a top surface and aplurality of wells, wherein each of the plurality of wells comprises afirst electrode disposed on the bottom of the well and a linker attachedto the sides of the well; and (b) a fluidic chamber system disposed onthe top surface of the substrate.

In one embodiment, the distance between a first well and a second wellis about 1 to about 200 μm. In another embodiment, the distance betweena first well and a second well is about 1 to about 10 μm.

In one embodiment, the substrate is quartz. In other embodiments, thesubstrate is selected from glass, sputtered SiO₂, PECVD, SiO₂ and thelike.

In one embodiment, the depth of the well is from about 500 nm to 500 μm.In another embodiment, the depth of the well is from about 500 nm to 10μm.

In one embodiment, the first electrode comprises palladium. In anotherembodiment, the first electrode comprises a material selected from, butnot limited to, gold, iridium, palladium, platinum or carbon.

In one embodiment, the linker comprises a hydroxy functionalized silane.In one aspect of this embodiment, the linker is(N-(3-triethoxysilylpropyl)-4-hydroxybutyramide. In another embodiment,the linker comprises an amino or thiol functionalized silane. In oneaspect of this embodiment, the linker is (3-aminopropyl)triethoxysilane(APTES).

In one embodiment, the fluidic chamber system comprisespolydimethylsiloxane (PDMS). In another embodiment, the fluidic chambersystem comprises PTFE. In another embodiment, the fluidic chamber systemcomprises parylene-C.

In one embodiment, the fluid chamber system has a height from about 50μm to about 10 mm. In another embodiment, the fluid chamber system has aheight from about 100 μm to about 5 mm. In another embodiment, the fluidchamber system has a height from about 500 μm to about 2.5 mm. Inanother embodiment, the fluid chamber system has a height from about 900μm to about 2.0 mm.

In one embodiment, the fluid chamber system comprising a secondelectrode. In one aspect of this embodiment, the second electrode ispalladium. In another embodiment, the second electrode comprises amaterial selected from gold, iridium, palladium, platinum or carbon.

In one embodiment, the fluid chamber system further comprises a systemfor introducing and removing liquids from the well.

FIG. 1A shows an apparatus design according to an embodiment of thedisclosure. The apparatus 1 comprises 4 counter electrodes 2 away fromthe reaction sites and 64 individually controlled electrodes 3.represents an integrated working electrode. FIG. 1B shows an array ofindividually addressable reaction sites connected to an external pad.FIG. 1C shows an exploded view of the reaction sites in which 4 is anelectrode, 5 is the substrate, 6 is the distance between reaction sitesand 7 is the depth of the reaction site. The DNA sequence is synthesizedon the functionalized side wall of reaction site.

The distance 6 between reaction sites are limited only by the diffusionlength of proton. The distance 6 is from about 1 μm to about 100 μm. Insome embodiments, the distance 6 is from about 30 μm to about 70 μm.

FIG. 2A-FIG. 2B show an apparatus design according to a secondembodiment of the disclosure. This apparatus is configured so that onlythe reactions sites are exposed, the effect of which is to minimizeunwanted reactions. As shown in FIG. 2C, a photo resist 9 is initiallyapplied to the substrate 5 and into the well, covering the firstelectrode 4. At reaction sites, the photo resist 9 is removed from thewell, exposing the first electrode 4 (FIG. 2D).

Method of Preparing Apparatus

The present disclosure also provides a method for preparing anapparatus. The method comprises (a) forming a plurality of wells on asubstrate; (b) applying a first electrode to the bottom of the well; (c)attaching a linker to the sides of the well; and (d) affixing a fluidicchamber onto the substrate.

In one embodiment, step (a) comprises: (i) applying a photo resist ontothe surface of the substrate to define the plurality of wells; and (ii)etching the substrate to create the plurality of wells.

In one embodiment, the substrate is silicon dioxide, quartz is used inone of the apparatus. In another embodiment, the substrate is selectedfrom glass, sputtered SiO₂, PECVD, SiO₂ and the like.

In one embodiment, the first electrode is palladium. In anotherembodiment, the first electrode comprises a material selected from, butnot limited to, gold, iridium, palladium, platinum or carbon.

In one embodiment, step (b) comprises thermally evaporating palladium.In another embodiment, step (b) comprises a material selected from gold,iridium, palladium, platinum or carbon.

In one embodiment, step (b) further comprises removing the photo resist.

In one embodiment, step (c) comprises immersing the substrate from step(b) into a linker solution for a period of time; and removing the linkersolution. In some aspects of this embodiment, step (c) comprises bakingat about 100 to about 150° C. for about 0.1 hr to about 2 hr.

In one embodiment, the fluid chamber system comprises a secondelectrode.

In one embodiment, the second electrode is palladium. In anotherembodiment, the second electrode comprises a material selected from, butnot limited to, gold, iridium, palladium, platinum or carbon.

In one embodiment, the fluid chamber system further comprises a systemfor introducing and removing liquids from the well.

FIG. 3A-FIG. 3D show a first method of preparing an apparatus asdescribed herein. In FIG. 3A, a photo resist 7 is applied onto thesurface of a substrate 5 to define a well. The substrate 5 is etched tocreate a well having a defined depth (FIG. 3B). A first electrode 4 isapplied to the bottom well, as shown in FIG. 3C. The photo resist 7 isthen removed, as shown in FIG. 3D.

FIG. 4A-FIG. 4E show a method of preparing an apparatus according to asecond embodiment of the disclosure. A Cr passivation layer 8 isthermally evaporated on a piranha cleaned quartz substrate 5 (FIG. 4A).A photo resist is spin coated and exposed to define a well (FIG. 4B). Cris removed by Cr etchant. RIE etching is used to create a well structurewith a defined depth (FIG. 4C). A palladium electrode is thermallyevaporated onto the bottom of the well (FIG. 4D). Then, the photo resistis removed, as shown in FIG. 4E.

FIG. 5A-5C show a method of preparing an apparatus according to a thirdembodiment of the disclosure. As shown in FIG. 5A, a photo resist 9,such as SU-8 2002, is spin coated to the substrate 5 and into thetrenches covering the first electrode 4. The photo resist passivationlayer pattern is exposed and post-baked before development to yield apassivation layer that seals all trenches with metal connections (FIG.5B), except for the reaction cite areas as marked in FIG. 2D. Thepassivation layer is finalized by a hard-baking procedure (FIG. 5C).

FIG. 6 shows the method for functionalizing the substrate with a linker10. The apparatus is initially cleaned with UV/ozone. The apparatus isthen immersed in a solution comprising the linker 10. In the immersionstep, the linker 10 attaches to the sides of the wells. In oneembodiment, the linker solution comprises linker, water and ethanol. Inone embodiment, the linker isN-(3-triethoxysilylpropyl)-4-hydroxybutyramide. After immersion in thelinker solution, the apparatus is baked, e.g., at about 120° C. forabout 20 min.

After the linker is attached to the sides of the wells, the apparatus ismodified to contain a fluidic chamber system. As shown in FIG. 7, a PDMSchamber 11 is fixed onto the apparatus surface. Then, chemical inlettubing 12 and outlet tubing 13 is fixed to the chamber. All chemicalsare delivered into the chamber through a fluidic multiplexer

Methods of Use

The present disclosure also provides a method of synthesizingoligonucleotides. The method comprises (a) providing an apparatus asherein described; (b) introducing a solution comprising a firstnucleoside phosphoramidite monomer and an activator into the well,wherein the first phosphoramidite monomer comprises a 5′-protectinggroup, an acid sensitive protecting group and optionally a basesensitive protecting group, and wherein the first phosphoramiditemonomer reacts with the linker attached to the side walls of the well toform a linked nucleoside through a phosphite triester; (c) removing thesolution from step (b) from the well; (d) introducing a solutioncomprising a capping reagent into the well, wherein the capping reagentreacts with any unreacted linker from step (b) to form a capped linker;(e) removing the solution from step (d) from the well; (f) introducing asolution comprising an oxidant into the well, wherein the oxidantconverts the phosphite triester of the linked nucleoside to a phosphatetriester; (g) removing the solution from step (f) from the well; (h)introducing a solution comprising a first deprotecting reagent into thewell, wherein the deprotecting reagent removes the 5′-protecting group;(i) removing the solution from step (h) from the well; (j) repeatingsteps (b) through (i) to synthesize a protected oligonucleotide; and (k)introducing a solution comprising a second deprotecting reagent into thewell, wherein the second deprotecting agent removes the protectinggroups on the oligonucleotide.

In one embodiment, the activator is 4,5-Dicyanoimidazole (DCI).

In one embodiment, the capping reagent is acetic anhydride. In oneembodiment, the capping solution comprises acetic anhydride,dimethylaminopyridine, 2,6-lutidine and THF.

In one embodiment, the capped linker comprises an acetate group.

In one embodiment, oxidant is (1S)-(+)-(10-camphorsulfonyl)-oxaziridine(CSO). In one embodiment the oxidant solution comprises CSO andacetonitrile.

In one embodiment, the first deprotecting reagent electrochemicallyremoves a 5′-trityl group. In one embodiment, the first deprotectingreagent is electrochemically generated acid. In one aspect of thisembodiment, the localization of the pH change by electrochemicalreaction is controlled by the depth and dimension of the reaction sites,the composition of the pH buffer solution above the reaction sites, andthe duration of the electric bias applied at the electrodes. In oneaspect, the pH buffer contains proton quencher 2,6 lutidine.

In one embodiment, the first deprotecting reagent is hydroquinone and2-6 lutidine. In one embodiment, the first deprotecting reagent solutioncomprises hydroquinone, anthraquinone, tetraethylammonium p-toluenesulfonate and acetonitrile. In one embodiment, when the firstdeprotecting reagent solution is introduced, a potential is applied tothe working electrode. In one embodiment, the potential is from about 1Vto about 5V. In another embodiment, the potential is from about 2V toabout 4V.

In another embodiment, the first deprotecting reagent is dichloroaceticacid. In one embodiment, the first deprotecting reagent solutioncomprises dichloroacetic acid in dichloromethane.

In one embodiment, the second deprotecting reagent removes the phosphateprotecting group.

In one embodiment, the second deprotecting reagent removes thenucleobase protecting groups.

In one embodiment, the second deprotecting reagent is ethylenediamine.In one embodiment, the second deprotecting reagent solution comprisesethylenediamine and ethanol.

As shown in FIG. 8 and FIGS. 9A-9H, the linker attached to the sidewells is functionalized by immersing the device into linker solution forabout 5 minutes to about 180 minutes and then baking for about 1 min toabout 180 minutes. In the second step, a solution comprising a firstnucleoside phosphoramidite monomer and an activator are introduced intothe well (FIG. 9A). The solution is flowed into the well from about 1sec to about 15 min at a volume from about 1 μL to about 5 mL. Inpreferred embodiment, the solution is flowed into the well from about 1sec to 1 min. In another preferred embodiment, the solution is flowedinto the well for about 30 sec.

The solution from the second step is removed and then, in the thirdstep, a solution comprising a capping reagent is introduced into thewell (FIG. 9B). The solution is flowed into the well from about 1 sec toabout 15 min at a volume from about 1 μL to about 5 mL. In preferredembodiment, the solution is flowed into the well from about 1 sec to 1min. In another preferred embodiment, the solution is flowed into thewell for about 15 sec. The capping reagent reacts with any unreactedlinker from the second step to form a capped linker. The solutioncomprising the capping reagent is removed from the well.

In the fourth step, a solution comprising an oxidant is introduced intothe well (FIG. 9C). The solution is flowed into the well from about 1sec to about 15 min at a volume from about 1 μL to about 5 mL. Inpreferred embodiment, the solution is flowed into the well from about 1sec to 5 min. In another preferred embodiment, the solution is flowedinto the well for about 3 min. The oxidant converts the phosphitetriester of the linked nucleoside to a phosphate trimester. The oxidantsolution is then removed from the well.

A solution comprising a first deprotecting reagent is introduced intothe well in the fifth step (FIG. 9D).

The solution is flowed into the well from about 1 sec to about 20 min ata volume from about 1 μL to about 5 mL with a potential applied to theworking electrode. In preferred embodiment, the solution is flowed intothe well from about 1 sec to 5 min. In another preferred embodiment, thesolution is flowed into the well for about 3 min. The deprotectingreagent removes the 5′-protecting group. The deprotection reagentsolution is removed and steps 1 through step 5 can be repeated aplurality of times as desired (FIG. 9E-FIG. 9G).

In the sixth step, a solution comprising a second deprotecting reagentis introduced into the well (FIG. 9H). The second deprotecting agentremoves the protecting groups on the oligonucleotide. The solution isflowed into the well from about 1 sec to about 20 min at a volume fromabout 1 μL to about 5 mL. In preferred embodiment, the solution isflowed into the well from about 5 min to 15 min. In another preferredembodiment, the solution is flowed into the well for about 10 min.

In order that this invention be more fully understood, the followingexamples are set forth. These examples are for the purpose ofillustration only and are not to be construed as limiting the scope ofthe invention in any way.

EXAMPLES Example 1

The objective of Example 1 was to demonstrate the ability of control DNAsynthesis electrochemically. The apparatus used in Example 1 is shown inFIG. 10A. Palladium electrodes are deposit on quartz surface throughelectron-beam evaporation.

A two-cycle DNA synthesis was conducted in three main steps. In step 1,a dT-CE Phosphoramidite was attached to the functionalized silicondioxide surface. In step 2, a 3.6V electric potential was applied toselected electrodes for 90 second to oxidize hydroquinone and theelectrochemically generated proton deprotected the first nucleotide onthe silicon dioxide surface. In step 3, a fluorescein labeled dT-CEphosphoramidite was coupled to the deprotected first nucleotide.

One positive control device and one negative control device were testedto compare the results. All three apparatuses were fabricated and testedunder same controlled condition.

To evaluate the nonspecific binding of the fluorescent labelednucleotide, during the detritylation step, the negative control devicewas flowed with hydroquinone deprotection solution for 10 minuteswithout electric activation. A 4 mW/488 nm laser was used forepi-fluorescence imaging light source.

To have a successful 2 cycle synthesis result to compare with, apositive control test was taken. During the detritylation step, astandard chemical deprotection (dichloroacetic acid deprotectionsolution) was performed.

Based on the synthesis result of the negative control, the fluorescentintensity measured (FIG. 10B) is no more than 800 (arbitrary unit). Asfor the synthesis result of the positive control, the fluorescentintensity measured (FIG. 11B) is about 4500. Taking the nonspecificbinding fluorescent signal as noise, the signal/noise ratio of thepositive control is around 5.6.

The synthesis result from the electrochemical deprotection sample isshown in FIG. 12A. The fluorescent intensity measured (FIG. 12B) isaround 4000 and the signal/noise ratio calculated is around 5, which iscomparable to positive control.

These results show the successful demonstration of the ability toconduct DNA synthesis with electrochemical control. The yield/intensityof the apparatus was comparable with standard chemical controlledsynthesis.

Example 2

The objective of Example 2 was to demonstrate the ability to conductlocalized electrochemical control of DNA synthesis and the diffusioncontrol of electrochemically generated proton in the apparatus. Theapparatus used in Example 2 is shown in FIG. 13. Palladium electrodesare deposit on quartz surface through electron-beam evaporation.

A two-cycle DNA synthesis was conducted in three main steps. In step 1,a dT-CE phosphoramidite was attached to the functionalized silicondioxide surface. In step 2, an electric potential was applied toselected electrodes to oxidize hydroquinone and the electrochemicallygenerated proton deprotected the first nucleotide on the silicon dioxidesurface. Three activation time were selected: 1 second, 5 second, and 10second. In step 3, a fluorescein labeled dT-CE Phosphoramidite wascoupled to the deprotected first nucleotide.

By the fact that fluorescent signal comes the second fluorescein labelednucleotide, the fluorescent intensity was used as an indicator of theaccessibility to electrical generated proton of the first nucleotide.The synthesis result shows a proportional increase of fluorescentintensity with electrode activation time. When the electrode isactivated for 1 second (FIG. 14A), the highest fluorescent intensityaround the trench is around 1800 (arbitrary unit) (FIG. 14B); for5-second activation electrode (FIG. 15A), the fluorescent intensity isaround 3500 (FIG. 15B); as for 10-second activation electrode (FIG.16A), the fluorescent intensity is around 5000 (FIG. 16B). The synthesisresult also indicates the proton diffusion length (the distance for thehighest fluorescent intensity decades to background). For 1 secondactivation, the longest distance the electrochemical generated protoncan travel is around 100 μm; for 5 second activation, the protondiffusion length is around 180 um; and for 10 second activation, theproton diffusion length is around 240 μm. These analyses confirmed thatthe apparatus conducts a localized DNA synthesis. Also, with longeractivation time, more proton is generated and hence introduced a highersynthesis yield (fluorescent intensity), but also allowed longerdistance for proton to diffuse thus the deprotection was less localized.By adding 2,6 lutidine as a proton quencher, the proton generated aroundthe electrode is neutralized by basic lutidine and hence reduce theproton diffuse distance.

Example 3

In this Example, a 12 mer DNA was synthesized through 12 secelectrochemical activation. The sequence was verified by hybridizationwith a fluorescent labeled complimentary DNA. Only the DNA with correctsequence shall hybridize with the fluorescent target and emitfluorescent signal. The result was compared with the hybridization of anidentical sequence synthesized through conventional chemical activation.

The device comprises a chromium passivation top surface, 1 μm SiO2 wellsetched by RIE, and 100 nm palladium electrodes deposit at the bottom ofthe wells.

The SiO₂ surface was cleaned by 3 min BHF etching before linkerfunctionalization for improved surface quality.

A 12 mer DNA (3′-TTT(spacer) TTA CGG TCA TAG GTC-5′) was synthesizedthrough 12 sec electrochemical activation. The purchased fluorescentlabeled DNA target (5′-AAT GCC AGT ATC CAG GTC GG/3FluorT/-3′) iscomplimentary to the 12 mer DNA synthesized. FIG. 17 illustrates thesuccessful hybridization between synthesized DNA and fluorescent labeledcomplimentary target.

The hybridization protocol was as follows:

(1) Buffer Preparation

Buffer (PB buffer): 100 mM NaCl, 10 mM Phosphate, pH 7.4

To prepare 100 mL of Phosphate Buffer:

-   -   Prepare 80 mL of distilled water in a volumetric flask.    -   Add 0.107 g of Sodium phosphate dibasic to the solution.    -   Add 0.0296 g of Sodium phosphate monobasic to the solution.    -   Adjust solution to final desired pH using HCl or NaOH    -   Add distilled water until volume is 100 mL    -   Add 0.5844 g NaCl

(2) DNA Target Solution

Making 70 μM aliquot DNA target solution

-   -   Add 1 mL of the buffer to dissolve a custom synthesized DNA        target    -   Divide the stock solution into 10 aliquots, store at −20° C.

(3) Dilute 70 μM target DNA solution into 1 μM target solution(determined by UV spectrometry))

-   -   Add 100 uL of 70 μM target solution to 6.9 mL of buffer for 7 mL        1 uM DNA target solution

(4) Heat up target solution to 95° C. for 5 min to remove any possiblesecondary structures

(5) Freeze the target solution in ice water for 5 min

(6) Immediately perform hybridization with synthesized probe forovernight

Heat up the chip to 75° C. and slowly cool down at a 1-5 ° C./min rate

(7) Brief rinsing with PBS

(8) N₂ dry and imaging

By the fact that fluorescent signal comes the second fluorescein labelednucleotide, fluorescent signal can be only observed when the DNA withcorrect sequence successfully hybridize with the fluorescent target.FIG. 18A shows the fluorescent optical image of an electrochemicallyactivated electrode and FIG. 18B shows the fluorescent intensitymeasurement across the reaction site. The fluorescent intensity measured(FIG. 18B) is about 300. Taking the background signal from the CCDdetector as noise (150), the signal/noise ratio of the positive controlis around 2 indicating successful synthesis of correct DNA synthesis.FIG. 18C and FIG. 18C show the fluorescent measurement result of anotheractivated site with signal/noise ratio around 2. The signal/noise ratiobetween activated sites are comparable. FIG. 19A and FIG. 19B show thefluorescent measurement result of a site without activation on the samedevice as negative control. No obvious fluorescent signal is observedwhich indicates no complete DNA product is synthesized at this reactionsite. FIG. 20A and FIG. 20B show the fluorescent measurement result of apositive control which the DNA sequence is synthesized throughconventional chemical activation with signal/noise ratio around 1.6which is comparable to electrochemical activation.

This example shows that electrochemical controlled 12 mer DNA synthesiswas successfully verified by the hybridization test.

Materials and Methods

Device Fabrication:

Quartz microscope coverslips (Electron Microscopy Sciences) were cleanedin hot 1:3 H₂O₂:H₂SO₄ solution (piranha clean) for 30 min, rinsed withmilli-Q water. Three layers of photo resist (HMDS/LOR 3A/AZ3312) werespin coated at 4000 rpm for 40 seconds. The trench pattern was thendefined with a GCA aligner and standard development procedure. Next, a20-minute wet etch was applied with Ultra Etch 20:1 (KMG) to create 800nm to 1000 nm wells. The exposed surface was cleaned by O₂ plasma for 1minute and then 100 nm palladium was deposited at the bottom of thewells with electron beam evaporation method. The lift-off process wasconducted by immersion in hot PG remover at 80° C. for 3 hr.

SiO₂ Surface Silanization:

The SiO₂ surface was first cleaned with O₂ plasma for 1 minute andimmediately immersed into linker solution (1.5%N-(3-triethoxysilylpropyl)-4-hydroxybutyramide, 5% water in ethanol) for1.5 hour, rinsed with ethanol, and baked at 115 C in an oven for 20minute.

Fluidic System

The apparatus itself and a PDMS chamber compose the fluidic system. Theapparatus was first adhered on a PCB board with PMMA. The selectedelectrodes for electrochemical reaction were bonded with a wedge-bondingmachine; a PDMS chamber was molded in a 3D printed mold and fixed on thedevice with silicon gel and a mechanical clamp. An inlet and an outletwere attached to top of the chamber for reagent delivery. All chemicalsneeded for the synthesis were delivered to the chamber through apositive pressure with a syringe. All reactions were done in a glove boxfor controlled atmosphere.

DNA Synthesis:

In this example, standard phosphoramadite chemistry was used for DNAsynthesis. Solution comprising the first nucleotide (2 mM dT, 2 mM DCIactivator in acetonitrile) was flowed through the camber for 10 minutesflowed by an acetonitrile flush; SCO oxidizer was then flowed throughthe chamber for 10 minutes followed by an acetonitrile flush; cappingmix was the next reagent flowed through with a 10 minute reaction timeand an acetonitrile flush was taken to flush all the residual chemicals.Next, for electrochemical detritylation/deblocking of the firstnucleotide, the deblocking solution (50 mM hydroquinone/2.5 mManthraquinone/0.1M tetraethylammonium p-toluenesulfonate) were flowedthrough the camber. A 3.6V potential was applied to selected electrodefor electrochemical activation with a battery. The same procedure wasfollowed for next cycle. At the end of synthesis, a deprotectingsolution of ethylenediamine in ethanol (1:1) was flowed through thechamber for 5 min. Ethanol and milli-Q water flush were performedsequentially and the device was stored in dark for imaging.

Imaging:

The synthesis result was analyzed with an epi-fluorescent microscope. A488 nm laser was used for excitation source. Laser power was set at 5 mWand exposure time was 500 ms. The fluorescent intensity was analyzedwith Nikon NIS-Elements software.

While particular materials, formulations, operational sequences, processparameters, and end products have been set forth to describe andexemplify this invention, they are not intended to be limiting. Rather,it should be noted by those ordinarily skilled in the art that thewritten disclosures are exemplary only and that various otheralternatives, adaptations, and modifications may be made within thescope of the present invention. Accordingly, the present invention isnot limited to the specific embodiments illustrated herein, but islimited only by the following claims.

1. An apparatus for synthesizing a biopolymer, the apparatus comprising:(a) a substrate comprising a passivized top surface and a plurality ofwells, wherein each of the plurality of wells comprises a firstelectrode disposed on the bottom of the well and a linker attached tothe sides of the well; and (b) a fluidic chamber system disposed on thetop surface of the substrate.
 2. The apparatus according to claim 1,wherein the distance between a first well and a second well is about 1to about 500 μm.
 3. The apparatus according to claim 1, wherein thediameter of the well is about 1 to about 200 μm.
 4. The apparatusaccording to claim 1, wherein the substrate is selected from glass,sputtered SiO₂, PECVD, SiO₂ and the like.
 5. The apparatus according toclaim 1, wherein the depth of the well is from about 500 nm to about 500um.
 6. The apparatus according to claim 1, wherein the depth of the wellis from about 1 to 10 μm.
 7. The apparatus according to claim 1, whereinthe first electrode comprises a material selected from gold, iridium,palladium or platinum.
 8. The apparatus according to claim 1, whereinthe linker comprises a hydroxy functionalized silane.
 9. The apparatusaccording to claim 1, wherein the linker is(N-(3-triethoxysilylpropyl)-4-hydroxybutyramide.
 10. The apparatusaccording to claim 1, wherein the fluidic chamber system comprisespolydimethylsiloxane (PDMS).
 11. The apparatus according to claim 1,wherein the fluid chamber system has a height range from 50 μm to 10 mm.12. The apparatus according to claim 1, wherein the fluid chamber systemfurther comprises a system for introducing and removing liquids from thewell.
 13. A method for preparing an apparatus, comprising a. forming aplurality of wells on a substrate; b. applying a first electrode to thebottom of the well; c. passivating the connections of the electrodes; d.attaching a linker to the sides of the well; and e. affixing a fluidicchamber onto the substrate.
 14. The method of claim 13, wherein step (a)comprises: i. applying a photo resist onto the surface of the substrateto define the plurality of wells; and ii. etching the substrate tocreate the plurality of wells.
 15. The method of claim 14, wherein thesubstrate is selected from glass, sputtered SiO₂, PECVD, SiO₂ and thelike.
 16. The method of claim 14, wherein the first electrode is amaterial selected from gold, iridium, palladium, platinum or carbon. 17.The method of claim 14, wherein step (b) comprises physical vapordeposition of material selected from gold, iridium, palladium, platinumor carbon.
 18. The method of claim 15, wherein step (b) furthercomprises removing the photo resist.
 19. The method of claim 14, whereinstep (c) comprises immersing the substrate from step (b) into a linkersolution for a period of time; and removing the linker solution.
 20. Themethod of claim 14, wherein the fluid chamber system further comprises asystem for introducing and removing liquids from the well.
 21. A methodof synthesizing an oligonucleotide, the method comprising (a) providingan apparatus, the apparatus comprising: (i) a substrate comprising apassivized top surface and a plurality of wells, wherein each of theplurality of wells comprises a first electrode disposed on the bottom ofthe well and a linker attached to the sides of the well; and (ii) afluidic chamber system disposed on the top surface of the substrate; (b)introducing a solution comprising a first nucleoside phosphoramiditemonomer and an activator into the well, wherein the firstphosphoramidite monomer comprises a 5′-protecting group, an acidsensitive protecting group and optionally a base sensitive protectinggroup, and wherein the first phosphoramidite monomer reacts with thelinker attached to the side walls of the well to form a linkednucleoside through a phosphite triester; (c) removing the solution fromstep (b) from the well; (d) introducing a solution comprising a cappingreagent into the well, wherein the capping reagent reacts with anyunreacted linker from step (b) to form a capped linker; (e) removing thesolution from step (d) from the well; (f) introducing a solutioncomprising an oxidant into the well, wherein the oxidant converts thephosphite triester of the linked nucleoside to a phosphate triester; (g)removing the solution from step (f) from the well; (h) introducing asolution comprising a first deprotecting reagent into the well, whereinthe electrochemically generated proton in the first deprotecting reagentremoves the 5′-protecting group; (i) removing the solution from step (h)from the well; (j) repeating steps (b) through (i) to synthesize aprotected oligonucleotide; and (k) introducing a solution comprising asecond deprotecting reagent into the well, wherein the seconddeprotecting agent removes the protecting groups on the oligonucleotide.22. The method of claim 21, wherein the capping reagent is aceticanhydride.
 23. The method of claim 21, wherein the capped linkercomprises an acetate group.
 24. The method of claim 21, wherein theoxidant is (1S)-(+)-(10-camphorsulfonyl)-oxaziridine (CSO).
 25. Themethod of claim 21, wherein the first deprotecting reagent removes a5′-dimethyltrityl group.
 26. The method of claim 25, wherein the firstdeprotecting reagent is hydroquinone.
 27. The method of claim 21,wherein the second deprotecting reagent removes the phosphate protectinggroup.
 28. The method of claim 21, wherein the second deprotectingreagent removes the base protecting groups.
 29. The method of claim 27,wherein the second deprotecting reagent is ethylenediamine.
 30. Themethod of claim 28, wherein the second deprotecting reagent isethylenediamine.